DESCRIPTION (applicant's abstract): There is an important need to develop accurate, simple and economic methods to determine total homocysteine (tHCY) levels in order to make such an assay a recognized part of standard medical practice available for the general population. HPLC methods for tHCY measurement have been developed and have been used as the standard assay for tHCY. HPLC is highly specialized and low throughput, however. A fluorescence polarization immunoassay for tHCY has also been developed. However, it appears that this method can only be practiced with specialized lowthroughput instrumentation. The currently used assays are therefore neither suitable for high-throughput tHCY measurement and nor for routine clinical laboratories. In Phase I, a simple high specificity and sensitivity tHCY enzymatic assay was developed using a homocysteine-speciflc recombinant homocysteinase (rHCYase) and H2Sspecific chromogenic agent N, N-dibutylphenylenediamine (DBPDA). The tHCY enzymatic assay highly correlates to the standard HPLC tHCY assay. The goal of Phase II is to apply the total homocysteine (tHCY) enzymatic assay developed in Phase I for broad base clinical use to enable tHCY to be a routine test as a risk factor for cardiovascular and other diseases. In order to achieve this specific goal, the specific aims of the Phase II application are to adapt the tHCY enzymatic assay on examples of widely-used instruments, a robotic microtiter plate reader and an automatic chemistry analyzer adapted for high throughput screening and routine testing. To adapt the tHCY enzymatic assay for robotic rnicrotiter plate readers, the Tecan Genesis (100/8) Robotic Sampler Processor will be used. To adapt the tHCY enzymatic assay for automatic chemistry analyzers, the Hitachi 912 automated chemistry analyzer will be used. Validation of the robotic microtiter plate reader and automatic chemistry analyzer tHCY enzymatic assays will be carried out by comparing their performance with the manual tHCY enzymatic assay thus far developed and the HPLC tHCY assay in a prospective clinical trial of the efficacy of high-dose folic acid to lower tHCY levels and improve outcome of patients having both end stage renal disease and cardiovascular disease. The tHCY enzymatic kits for these applications will be ready for commercial launch at this point. PROPOSED COMMERCIAL APPLICATION: Not Available